PROJECT SUMMARY Better therapies and diagnostic approaches are urgently needed to improve patient outcomes in sepsis. In this condition, excessive inflammation, followed by general immunosuppression, contributes to organ failure, opportunistic infections, and often, death. Given the complexity of the disease, a multifaceted approach is necessary. Our long-term goal is to explore the diagnostic and therapeutic potential of extracellular vesicles (EVs) during sepsis. EVs are small, membrane-derived vesicles that function in intercellular communication. EVs are elevated in the blood during sepsis and induce coagulation and inflammation; however the mechanism by which they are produced and cause inflammation is not fully understood. The overall objective of this application is to characterize EV biogenesis during bacterial infection and define the role of EVs and EV-associated DNA in inflammation and innate immunity. It was previously shown that S. aureus triggers release of bacteriostatic EVs from neutrophils, and our preliminary data shows that these EVs, which associate with both human and bacterial extravesicular DNA and whole bacteria, potentiate the inflammatory response in a DNA-dependent fashion. On the basis of these data, we will test the hypothesis that EVs serve as a delivery vehicle for extracellular DNA, and that EVs and DNA work in concert to promote inflammation and alter cellular functions. To test this hypothesis, we propose the following specific aims: (1) Identify the pathway of EV release and characterize the interaction of EVs with extracellular DNA and bacteria. Completion of this aim will contribute to our understanding of EV biogenesis and will lead to the identification of the specific interactions, as well as the processes, that could be targeted therapeutically to limit deleterious outcomes in sepsis. Additionally, comparisons of EVs produced in response to S. aureus or other bacteria that commonly cause sepsis will be performed to determine if the association of extravesicular DNA with EVs could be exploited as a diagnostic tool. (2) Test the hypothesis that TLR9 detects EV-associated bacterial DNA and triggers pro-inflammatory cytokine production. Completion of this aim will establish whether bacterial DNA associated with EVs directly causes inflammation and will determine the mechanism underlying extracellular DNA sensing by pattern recognition receptors. (3) Examine whether elimination of both DNA and bacteria is able to shift the role of EVs from pathogenic to protective. Limiting inflammation would be beneficial in the early progression of sepsis; however, prevention of opportunistic infections is equally important. By completing this aim, we will determine whether EVs enhance macrophage effector functions, and whether DNase and antibiotic treatment of EVs effectively shifts EVs from pro- inflammatory to immunomodulatory. This work is innovative and significant, in that pinpointing mechanisms by which EVs are generated and the ways in which they are able to modulate inflammation and effector functions will contribute to a deeper understanding of the role of EVs in innate immunity. These results will ultimately enhance our ability to exploit EVs as a diagnostic tool and therapeutic going forward.